1. Field of the Invention
The present invention relates to a method for classifying and counting leukocytes, more particularly a method for classifying and counting leukocytes which allows immature granulocytes different in degree of maturity to be classified and counted by a flow cytometry.
2. Description of Related Art
In the field of clinical examination, classifying and countering immature granulocytes according to the degree of maturity is advantageous since it provides significantly useful information for making a correct diagnosis of a disease and for observing the course of a disease.
For example, immature granulocytes normally exist in bone marrow but do not exist in peripheral blood. For this reason, the presence of immature granulocytes in peripheral blood of a patient indicates the possibility that the patient has an infectious disease, inflammatory disease, chronic or acute myelocyticleukemia, malignant tumor or other disease. Accordingly, classifying and enumerating immature granulocytes different in the degree of maturity is helpful for diagnosing these diseases and for monitering the progress of the diseases.
Conventionally, a common method for classifying and counting immature granulocytes is comprised of preparing a smear sample of blood, staining the sample with a suitable dye, microscopically classifying cells in the sample into categories of promyelocytes, myelocytes and metamyelocytes according to their degree of maturity, and counting cells in each category. However, this method requires a complicated pretreatment before microscopic observation. Accuracy of the method is poor because only a limited number of cells can be counted. Furthermore, considerable skill of the operator is required for accurate classification. Reproducibility is limited due to the small number of leukocytes enumerated.
On the other hand, a variety of methods utilizing the principle of flow cytometry have been proposed for analysis of blood components in marrow fluid which contains blood cells in various stages of maturity.
For example, U.S. Pat. No. 5,137,809 has proposed a method for identifying different cell series and stages of maturity of cells in marrow fluid using a first antibody for indicating leukocytes and a second antibody for classifying leukocytes into subpopulations. However, this method has a problem in that immature granulocytes cannot be classified according to their degree of maturity as by microscopic observation, because the stages of maturity are indicated only by one parameter, i.e., the second antibody.
Bowen et al have reported that the intensity of expression of CD 16 and CD11b reflects the degree of maturity of granulocytic cells in bone marrow (Abnormal patterns of expression of CD16(FcRgIII) and CD11b(CRIII) antigens by developing neutrophils in the bone marrow of patients with myelodysplastic syndrome (Laboratory Hematology, Vol.3, No.4, 292 to 298,1997)). According to this report, granulocytic cells are first indicated with CD45 and SSC (side scattered light) and the degree of maturity of the granulocytic cells can be judged by checking the intensity of expression of CD16 and CD11b in the granulocytic cells. However, this method cannot distinguish mature eosinophils from immature granulocytes and therefore analysis results are poor. Particularly, both mature eosinophils and immature granulocytes are CD16-negative and CD11b-negative and cannot be separated from each other.
For analyzing leukocyte components in a sample of peripheral blood, a variety of methods have been proposed.
For example, U.S. Pat. No. 5,413,938 has proposed a method for measuring immature granulocytes by lysing mature leukocytes with a reagent containing a nonionic surfactant and an amino acid. However, according to this method, all leukocytes except immature granulocytes are lysed and thus cannot be enumerated. Therefore, this method alone cannot calculate the ratio of an immature granulocyte count to a leukocyte count.
Hūbl et al have reported a method for detecting immature neutrophils on the basis of expression of CD16 (Value of neutrophil CD16 expression for detection of left shift and acute-phase response; Am. J. Clin. Pathol., 107(2): 187-196, February 1997). However, this method does not describe a method for classifying immature granulocytes according to the degree of maturity.
Under these circumstances, there is a demand for a method for accurately counting immature granulocytes in peripheral blood independently of effect of other leukocytic cells and classifying the immature granulocytes according to their degree of maturity.